User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/29

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07/29/11 ✓ Cultures/ minipreps: KAH219-224/pSB31 (retrial, 2 each) ✓ Assemblies: KAH228 - 231 Minipreps > Check with E/P digests Reagent Volume Expected: pSB31 = 5127 1-3. KAH219/pSB31 = 2422 4-6. KAH220/pSB31 = 1345 7-9. KAH221 = 1645 10-12. KAH222 = 2614 13-15. KAH223 = 1537 16-18. KAH224 = 1837 15 μL/lane; 1% agarose DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 PstI 1.0 dH2O 9.5   15 μL --> 37°C/ ~15 min. --> None worked. Try again with more insert + adapter. Assemblies KAH228: KAH54/(S/P)/430 + (1) KAH226/(X/P)/1564 KAH229: hPCD/(S/P)/186 + " KAH230: (2) KAH227/(S/P)/577 + polyA2/(X/P)/225 > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) up to 25 μL 10x buffer 3.0 enzyme 1 1.0 enzyme 2 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH226 (X/P) 1.099 1.85 55.0 2. KAH227 (S/P) 0.257 1.88 12.9 > Ligations Ligation Plate results (lig : neg crtl) 07/30/11 1. KAH226(X/P)/1564, 17 ng + KAH54(S/P)/~3630, 20 ng KAH228 >10:1 (Pick 2) 2. KAH54(S/P)/ 20 ng   3. KAH226(X/P)/1564, 18 ng + hPCD(S/P)/~3386, 20 ng KAH229 >10:1 (Pick 2) 4. hPCD(S/P)/ 20 ng   5. polyA2(X/P)/225, 1 ng + KAH227(S/P)/~3777, 20 ng KAH230 >10:1 (Pick 2) 6. KAH227(S/P)/ 20 ng     1 2 3 4 5 6 Insert DNA 0.5 --- 0.5 --- 0.5 --- Vector DNA 1.0 1.0 0.3 0.3 1.5 1.5 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 dH2O 2.5 3.0 3.2 3.7 2.0 2.5   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL --> Add 30 μL DH5α Turbo; plate on 100 μg/mL Amp